Corneal Molecular and Cellular Biology for the Refractive Surgeon: The Critical Role of the Epithelial Basement Membrane

Abstract

Purpose: To provide an overview of the recent advances concerning the corneal molecular and cellular biology processes involved in the wound healing response after excimer laser surface ablation and LASIK surgery.

Methods: Literature review.

Results: The corneal wound healing response is a complex cascade of events that impacts the predictability and stability of keratorefractive surgical procedures such as photorefractive keratectomy and LASIK. The generation and persistence of corneal myofibroblasts (contractile cells with reduced transparency) arise from the interaction of cytokines and growth factors such as transforming growth factor beta and interleukin 1 produced by epithelial and stromal cells in response to the corneal injury. Myofibroblasts, and the opaque extracellular matrix they secrete into the stroma, disturb the precise distribution and spacing of collagen fibers related to corneal transparency and lead to the development of vision-limiting corneal opacity (haze). The intact epithelial basement membrane has a pivotal role as a structure that regulates corneal epithelial-stromal interactions. Thus, defective regeneration of the epithelial basement membrane after surgery, trauma, or infection leads to the development of stromal haze. The apoptotic process following laser stromal ablation, which is proportional to the level of attempted correction, leads to an early decrease in anterior keratocyte density and the diminished contribution of these non-epithelial cells of components such as perlecan and nidogen-2 required for normal regeneration of the epithelial basement membrane. Haze persists until late repair of the defective epithelial basement membrane.

Conclusions: Defective regeneration of the epithelial basement membrane has a critical role in determining whether a cornea heals with late haze after photorefractive keratectomy or with scarring at the flap edge in LASIK.

Figure 1.

Slit-lamp photographs of late corneal haze after refractive surgery. (A) Central corneal haze after high-correction PRK ablation. (B) Circumferential corneal haze in the flap edge of LASIK treatment. Magnification 20X.

Figure 1.

Slit-lamp photographs of late corneal haze after refractive surgery. (A) Central corneal haze after high-correction PRK ablation. (B) Circumferential corneal haze in the flap edge of LASIK treatment. Magnification 20X.

Figure 2.

Chimeric (bone marrow transplant from green fluorescent protein [GFP+] mouse) mouse cornea with haze at 1 month after irregular PTK (method used to produce haze in mice). In the overlay (A), a high concordance can be seen between the red stain for alpha-smooth muscle actin marker for myofibroblasts (B) and the green stain for GFP+ (C) in several cells in the anterior stroma (arrows). Blue is DAPI staining of nuclei. Magnification: 800X. Reprinted by permission from Barbosa et al. Corneal myofibroblast generation from bone marrow-derived cells. Exp. Eye Res., 2010; 91:92-6.

Figure 3.

Central cornea of rabbit eyes assayed for apoptotic cells by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). (A) Rabbit eye 4 hours after −4.5D PRK ablation. (B) Rabbit eye 4 hours after −9.0D PRK ablation. Arrowheads indicate corneal surface in each panel. Arrows indicate some of the apoptotic keratocytes. Magnification: 200X.

Figure 4.

Lacunae developing in the previously confluent haze of a human cornea at 2 years after high correction PRK performed without mitomycin C. Magnification: 30X.

Figure 5.

Transmission electron microscopy of sections from untreated and treated central corneas of rabbits. (A) A representative image from an untreated-control cornea. Regular pattern of lamina lucida and lamina densa (arrows) is clearly seen between the epithelium (e) and stroma (s). (B) Representative image of rabbit cornea at 1 month after −4.5D PRK with a regenerated epithelial basement membrane, presenting ultrastructure of lamina lucida and lamina densa (arrows) very similar to the control cornea in (A). (C) Representative image of rabbit cornea at 1 month after −9.0D PRK showing no lamina lucida-like or lamina densa-like structures suggestive of regenerated epithelial basement membrane. Also note the disorganized extracellular matrix (d) and myofibroblasts (m) with large amounts of rough endoplasmic reticulum in the stroma beneath the epithelium. Magnification: 30,000X.

Figure 6.

Immunohistochemistry for nidogen-2 in human corneas. (A) Nidogen-2 protein was noted in the epithelium (e) and epithelial basement membrane, as well as at low levels in keratocytes (arrows) in unwounded control corneas. (B) In a cornea at 30 minutes after epithelial scrape, nidogen-2 protein was up-regulated in stromal keratocytes (arrows), including anterior stromal keratocytes in the early stages of apoptosis detected with the TUNEL assay (not shown). Magnification: 400X. Reprinted by permission from Torricelli et al. Exp Eye Res., 2015;134:33-8.